The invention relates to DNA protein complexes, proteins, DNA sequences, and antibodies that are suitable for the detection of cells having an unlimited proliferation and tumor-formation potential; methods for obtaining such DNA protein complexes, proteins or DNA sequences and their use to identify animal and human cells having an unlimited proliferation and tumor-formation potential.
All differentiated human and animal cells have a limited in vivo and in vitro proliferation potential before they undergo aging (senescence) and cell death. The number of cell divisions that are possible at a given time depend on the degree of differentiation of a cell, its age, and the species of the donor from which the cell was obtained, and on the duration of the cell cultures (Goldstein, S.: Replicative Senescence, Science 249 (1990), 1129-1133). Unlimited proliferation, however, is frequently found in neoplastically transformed cells. They thus form constantly growing symplasms leading to the formation of a tumor and, finally, together with the altered characteristics of other properties of these cells to tumor diseases. As compared to benign tumors, these malignant tumors are clinically characterized by their rapid growth and frequent formation of metastases. A neoplastic transformation of the cell is characterized by a heterogeneous picture of numerous alterations of the cellular morphology and physiology which also depend on the degree of differentiation of the cells. To date, only few molecularly definable parameters of neoplastic transformation are known; they include, for example, an altered degree of methylation of certain genes (W. Doerfler et al., Eukaryotic DNA methylation: facts and problems, FEBS Letters 268 (1990), 329-330), modified gene expression, or altered phosphorylation of certain gene products.
What is common to most tumor cells is the capability of unlimited in-vivo proliferation. Based on various observation, investigators assume that the proliferation of tumor cells which is independent of growth factors is, indeed, a frequently occurring characterisitic of tumor-forming cells, but not an absolutely necessary one (M. Strauss, B. E. Griffin, Cellular Immortalization, Cancer Cells 2 (1990), 360-365). However, for many questions in the field of diagnostics and therapy, it is of critical importance to identify those cells which no longer obey normal proliferation control and, hence, have acquired the potential for unlimited proliferation.
The problem involved in this matter is to distinguish this property of unlimited proliferation of tumor cells from the capability to regenerate that is found in many tissues. Regeneration is a controlled, short (transient) cell replication and only a intermittent suppression of programmed cell death as a response to a physiological stimulation. The transient proliferation of normal cells is again inhibited after regeneration of the tissue by still unidentified signals.
Known methods for the detection of malignant tumor cells are based on clinical, histological, and cytological observations, and on altered physiological measurements. Based on histological examinations, diagnostic routine methods are usually done with tissue sections (W. A. D. Anderson and J. M. Kissane, Pathology I, II, Mosby, Saint Louis 1977, 7th edition; C. S. Herrington and J. O. D. McGee, Diagnostic Molecular Pathology, Oxford University Press, Vol. I, II, 1st ed., 1992). As opposed to benign growth, the group of malignant cells is only vaguely separated from adjacent tissue, and the cells grow into surrounding tissue which they infiltrate and destroy. Most cases are triggered by a perifocal inflammation. Frequently, a great number of mitoses indicate increased proliferation activities of the tumor cells.
In addition to these histological examinations, it has become more common to use antibodies to detect proliferating malignant cells which recognize antigens that are preferably expressed by proliferating tumor cells such as Ki67 (D. C. Matthews, F. O. Smith, I. D. Bernstein, Monoclonal antibodies in the study and therapy of hematopoietic cancers, Curr. Opinion Immunol. 4 (1992), 641-646; M. Schwouzen, V. Diehl, M. Pfreundschuh, Immunozytologische Phxc3xa4notypisierung von Leukxc3xa4mien und Lymphomen, Med. Klinik 85 (1990), 533-547). However, these methods are based on the determination of indirect parameters without detecting the molecular processes that are linked to unlimited proliferation.
It is, hence, an object of the invention to identify cells with a potential of unlimited proliferation, especially malignant tumor cells, in a rapid and reliable manner without cultivating these cells in vitro or propagating them after injection in laboratory animals. It should be possible to distinguish between cells having the potential for unlimited proliferation and cells with transient proliferation in regenerating tissue.
This object is accomplished with a DNA protein complex (hereinafter referred to as complex) which is suitable for detecting human or animal having an unlimited proliferation and tumor-formation potential. Said complex can be obtained by isolating a mitochondria-free fraction of the cytoplasm from human or animal cells which can permanently divide and have a density of approx. 1.82-1.89 g/cm3 in a cesium chloride gradient; this is followed by isolating the complex from this fraction by extraction with phenol and precipitation with ethanol.
Experience has surprisingly shown that cells having an unlimited proliferation and tumor-formation potential, as opposed to normal resting cells, senescent cells, or cells with transient proliferation do have such complexes in their cytoplasm. With these complexes in accordance with the invention it is possible to detect such cells, which exhibit unlimited growth potential as a consequence of malignantant growth. In order to accomplish this, one uses cytoplasm or a suitable cytoplasm fraction in a complex in accordance with the invention as a standard in a gel electrophoresis by determining the DNA contents or reaction with a specific antibody.
In order to isolate complexes of the invention, cytoplasts of human or animal cells that are permanently capable of dividing are obtained according to known methods (for example EP-B-0 093 436, EP-B-0 256 512 and Proc. Natl. Acad. Sci. 85 (1988), 468-472 and lysed for example using detergents like NP40 or SDS, Wigler and Weistein, Biochem. Biphys. Res. Commn. 63 (1975) 669-674). The mitochondria are separated from these cytoplast fractions, preferably with the aid of a sucrose gradient (J. Biol. Chem. 249 (1974) 7991-7995). Those fractions who do not contain any mitochondria are incubated with RNase, preferably RNase A and RNase T1 as well as with proteinase, e.g. proteinase K or pronase. A fraction from this mixture with a density of approx. 1.82-1.89 g/cm3 in a cesium chloride gradient is enriched, and from this fraction, a complex is isolated by extraction with phenol and precipitation with ethanol. A fraction which has a density of approx. 1.82-1.89 g/cm3 in a cesium chloride gradient can be obtained both by means of centrifugation with a cesium chloride density gradient and by means of electrophoretic separation; these methods are known to the expert in the field (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 2nd edition, 1989).
The mitochondria-free fraction of the cytoplasm that is used for isolating the complexes can also be obtained by repeated freezing and thawing of the cells or by obtaining a cell fraction which is free of nuclear DNA. Such a fraction is preferably obtained by lysis of the cell with sodium chloride and SDS (xe2x80x9cHirt extractionxe2x80x9d, J. Mol. Biol. 26 (1967) 365-369) followed by centrifugation. The complexes can then be isolated from the supernatant.
In a preferred method, the complexes are isolated via sedimentation from a cytoplasmatic lysate of the cells to be examined using a salt gradient. The cells are lysed in a Mg2+-containing buffer (50 mmol/l Tris-HCl, pH 7.2, 10 mmol/l EDTA, 3 mmol/l MgCl2) by repeated freezing and thawing without using a detergent; the nuclei are sedimented by centrifugation at 6000xc3x97g and the supernatant is digested with proteinase K (100 xcexcg/ml) for 1 h at 60xc2x0 C. The released complexes are isolated by centrifugation (150,000xc3x97g, 10 h) with the aid of a CsCl two-step gradient; the lower fraction has a density of approx. 1.80 g/cm3 while the upper fraction has a density of approx. 1.70 g/cm3. The cytoplasmic complexes will sediment while the chromosomal DNA or complexes without covalent DNA protein binding will remain in the supernatant.
This method allows a very rapid isolation of cytoplasmatic complexes utilizing the following properties:
The complexes are released by freezing and thawing a Mg2+-containing buffer without lysing the nucleic or using detergents.
The complexes are stable with respect to proteinases while cytoplasmic organelles and membranes are digested.
The complexes have a very high density (approx. 1.86 g/cm3) and can be separated from chromosomal or other cytoplasmic DNA with the aid of salt gradients.
The DNA protein binding is stable with respect to high salt concentrations.
The complexes can be obtained from cytoplasm of any transformed cell, particularly permanently growing human animal cells, or from tumor cells, especially those that were obtained from tumor biopsies. Transformed cells are understood to be those that are rendered immortal by adding an agent. Permanently proliferating cells of different species (e.g. human, mouse, rat) and cells of different degrees of differentiation (fibroma, myeloma, ascites cells) contain complexes which differ with respect to their protein and/or DNA contents. The complexes of the invention are, hence, specific with respect to species and degree of differentiation. For the detection of cells having an unlimited proliferation and tumor-formation potential, one isolates complexes from cells of identical species or cell types similar to the one to be examined. For the detection of mouse fibroblasts/fibroma cells or mouse ascites tumor cells with unlimited proliferation, it is particularly preferred to use complexes that were isolated from the cytoplasts of transformed mouse L cells (L929 cells, ATCC CCL 1) or Ehrlich ascites cells (ATC CL 77) according to described methods. In order to obtain complexes of permanently proliferating cells of other tissues or degree of differentiation as well cells of other species, it is also possible to use other tumor cell lines with the desired degree of differentiation and species as a source for isolating the complexes. When obtaining corresponding complexes from human cervix tumors, it is preferred to use the human cell lines HeLa, or from B-lymphoma lines and the B-cell lymphoma line BJAB. Moreover, it is also possible to use permanently growing cell lines that were immortalized as a consequence of an experimental immortalization, e.g. by fusion of primary cells with cytoplasts of permanently growing cells (EP-B-0 093 436, Abken et al., J. Cell. Biol. 130 (1986) 795-805) or by transfection with DNA from these cytoplasts (EP-B-0 256 512 and DE 42 18 945.4 and Abken et al., Proc. Natl. Acad. Sci. 85 (1988) 468-472).
Experience has shown that a cytoplasmic DNA fraction with a density of 1.86 g/cm3 prevails in Hodgkin lymphoma cells. This DNA is also linked to proteins. The DNA molecules contained in the fraction have a length between 50 and 500 bp. As is the case with cells from mouse tumors, all DNA molecules are linear. However, they do not hybridize with those of the mouse tumor cells. This demonstrates that both animal and human lymphoma cells in accordance with the invention contain cytoplasmic DNA sequences. The fraction in accordance with the invention can also be found in human tumor cells of colon and mamma carcinomas and human melanomas.
It is possible to isolate the proteins and/or the DNA from these complexes and to use these to detect human or animal cells having an unlimited proliferation and tumor-formation potential.
Another subject matter of the invention are, hence, proteins with a molecular weight of approx. 52, 62 and/or 64 kD which are suitable for the detection of human or animal cells having an unlimited proliferation or tumor-formation potential; they can be obtained by treating a complex of the invention with DNase I and isolating the released proteins of approx. 52, 62 and/or 64 kD and chromatographic or electrophoretic procedures.
In order to isolate these proteins, the complexes are treated with DNase I and the digested nucleic acids are separated from the proteins via gel filtration; the proteins are at the same time separated according to size. When using complexes from mouse L cells (L929) and/or Ehrlich ascites tumor cells, proteins with a size of 52, 62 and 64 kD are obtained. These proteins are characterized by their capability of binding the cloned pLC 108 DNA (SEQ ID NO 1) in accordance with the invention in the presence of 8 mM Zn2+. The proteins from the Ehrlich ascites cells are characterized by their capability of binding cloned pEFC38 DNA (SEQ ID NO 29) in the presence of 8 mM Na+. It is preferred to use a template-bound DNase for the preparation of these proteins, e.g. DNase that is covalently linked to sepharose.
Another subject matter of the invention are antibodies suitable for the detection of human or animal cells having an unlimited proliferation and tumor-formation potential; they can be obtained by immunization of animals with a complex or protein or DNA in accordance with the invention. It is possible to use any animal commonly used for immunization, including mice (e.g. MNRI or BALB/c strain) or rabbits and also immunization protocols (cf. e.g. J. Peters and H. Baumgarten, Monoklonale Antikxc3x6rper, Springer Verlag, 2nd edition 1988).
The immunization of complexes in accordance with the invention, DNA and proteins generates antibodies that specifically bind to proteins from the cytoplasm cells having an unlimited proliferation or tumor-formation potential, but not to proteins from cells with normal proliferation capacities. The detection of these proteins with the aid of antibodies in accordance with the invention can be accomplished with the aid of methods such as ELISA, fluorescence methods, immunoassays, and competitive immunoassays all of which are known to the expert.
In addition to the protein portion of the complexes in accordance with the invention, the DNA portion can also be used for the detection.
Another subject matter of the invention is, hence, a DNA suitable for detecting human or animal cells having an unlimited proliferation or tumor-formation potential which can be obtained by means of cloning or enzymatic replication of the DNA of a complex in accordance with the invention.
Experience has surprisingly shown that the DNA of a complex of the invention can be ligated into cloning vectors like isolated DNA or can be enzymatically amplified, e.g. in a PCR, although in the complexes, this DNA is so tightly bound to the protein that it cannot be removed neither by means of detergents, proteases, high salt concentrations nor by means of thermal treatment. The DNA can be obtained by ligating the DNA of the complexes, e.g. in common vectors such as pUC19 and cloned in host organisms that are known to the expert such as E. coli HB101 or E. coli DH5xcex1. Recombinant clones that contain the DNA sequence of the invention are then identified by hybridization with the complexes of the invention and the DNA is isolated. It was thus possible to obtain 20 recombinant plasmid clones from L929 cells (ATCC CCL 1) and 25 recombinant plasmid clones from Ehrlich ascites cells (ATCC CCL 77) which are suitable for the molecular identification of cells with an unlimited proliferation potential. These DNA sequences are listed in sequence protocols SEQ ID NO 1-45. With the aid of the sequences it is possible to detect other suitable DNA sequences of other cells of different differentiation degrees or other species in order to identify corresponding cells with an unlimited proliferation potential.
The DNA sequences shown in SEQ ID NO 1 and SEQ ID NO 29 are suitable for immortalization of human or animal cells (cf. German patent application P 42 18 945.4). All other DNA sequences (SEQ ID NO 2-28 and SEQ ID NO 30-45) have no immortalizing effect, but are suitable for detecting human or animal cells having an unlimited proliferation or tumor-formation potential.
A preferred subject of the invention is, hence, a DNA in accordance with the invention which contains one of the DNA sequences shown in SEQ ID NO 2-28 or SEQ ID NO 30-45 or hybridizes with one of these sequences.
Another preferred subject matter of the invention are DNA which contain one of the DNA sequences shown in SEQ ID NO 46-61 or hybridize with one or several of these sequences. The nucleic acid SEQ ID NO 46-58 are derived from DNA of HD 428 Hodgkin cells. The sequences of SEQ ID NO 59-61 are derived from DNA clones of MCF 7 cells (mamma carcinoma).
The nucleic acids of the invention can be DNA, but also RNA or nucleic acid analogs, e.g. those where the sugar phosphate back bone has been replaced by a polypeptide chain. The nucleic acid can be double-stranded, preferably, however, it is single-stranded. The invention also addresses each of the complementary sequences. In a preferred manner, the nucleic acids of the invention contain a sequence of at least 15 amino acids whereby the sequences are selected from a group of at least 15 amino acids taken from
a sequence contained in a sequence according to SEQ ID NO 2-28 or 30-61,
a sequence that is obtained by linking two sequences of SEQ ID NO 1-45, 46-58, or 59-61,
sequences which code for the same amino acid sequence as the above listed two types,
sequences which contain, within the specific sequence, at least 70%, preferably more than 90%, particularly preferred more than 95% of sequences that are specific to the above listed two types.
The nucleic acids have a particularly preferred length between 16 and 100 nucleotides. Depending on the length they can be obtained with the aid of molecular biological or chemical/synthetic methods.
Experience has surprisingly shown that most of the so far tested DNA sequences contain the consensus sequence NNAAANTNTNGAANTGTANNANTGNAA (SEQ ID NO 62).
Another subject matter of the invention is a method for obtaining the DNA in accordance with the invention by isolating a complex in accordance with the invention of human or animal cells that are capable of permanently dividing followed by cloning the DNA from this complex or replicating it enzymatically.
Another preferred subject matter of the invention is a method for obtaining a DNA in accordance with the invention where the DNA sequences in accordance with the invention are identified by hybridization of a genomic gene bank or a cDNA bank of human and animal cells with one of the DNA sequences shown in SEQ ID NO 1-61 and then isolating the hybridizing cloned DNA from the gene bank according to known methods. It is possible to use gene banks of permanently proliferating tumor cells and of normal cells or tissues.
Yet another preferred subject matter of the invention is a method for obtaining a DNA in accordance with the invention by binding the DNA of human and animal cells to a protein in accordance with the invention; the proteins are bound on nitrocellulose or other carrier membranes and incubated (e.g. for 4xc2x0 C. for 30 min) in the presence of Zn2+ or Na+. Subsequently, the mixture is stringently washed at a high salt concentration (e.g. 0.1% SDS, 2xc3x97SSC).
Another preferred subject matter of the invention is a method for obtaining a DNA in accordance with the invention by enzymatically replicating a DNA or RNA fraction of human or animal cells; the starter molecules used are oligonucleotide molecules which hybridize with one of the sequences shown in SEQ ID NO 1-61. Preferred oligonucleotide starter molecules are those with a length between 20 and 30 base pairs. Enzymatic replication of the desired DNA sequences with the aid of polymerase chain reaction (PCR) is carried out according to methods that are known to the expert (Mullis and Fallona, Meth. Enzymol. 155 (1987) 335-350, Saiki et al., Science 239 (1988) 487-491). It is possible to use both gene banks as well as DNA or RNA preparations or cell lysates of permanently proliferating or normal senescent cells of humans or animals as DNA fractions.
Another variant of the method in accordance with the invention is the chemical synthesis of DNA in accordance with the invention containing one of the DNA sequences shown in SEQ ID NO 1-61 or parts of this DNA sequence. The synthesis is carried out according to oligonucleotide synthesis methods that are commonly used by the expert (F. Eckstein, ed., Oligonucleotides and analogues: A practical approach, IRL Press at Oxford University press, Oxford (1991)); it is preferred to use commercially available oligonucleotide synthesis instruments.
Another subject matter of the invention is a method for the detection of human or animal cells having an unlimited proliferation or tumor-formation potential by detecting a complex in accordance with the invention in a mitochondria-free fraction of the cytoplasm of the cell to be examined. Normal cells with limited proliferation capacities do not contain such complexes in this fraction of the cytoplasm while cells with unlimited proliferation, e.g. tumor cells, contain several hundred copies of these complexes.
The complexes in accordance with the invention are excreted. The DNA-protein-link of these complexes is very stable against proteolytic digestion. The complexes can be detected in cell supernatants over a period of several weeks.
The complexes can also be contained in peripheral blood, in urine, or in other body fluids where they can be detected.
A preferred subject matter of the invention is, hence, a method for the detection of human or animal cells with unlimited proliferation or tumor-formation potential by detecting a complex in accordance with the invention in body fluids.
The complexes can be determined via their DNA contents or via gel electrophoresis while the complexes in accordance with the invention are carried along as standards for the determination of a positive reaction. In these methods, however, it is not possible to distinguish between complexes of different species or tissue types. Such a specific detection is, however, possible with the aid of the antibodies in accordance with the invention.
In addition to complete antibodies, it is also possible to use antibody fragments which contain the specific antigen recognition regions of the light and heavy chains (VL and VH). These fragments can be coupled to carriers such as proteins, sugars or sepharose, or can be linked to dyes to generate fusion products.
Such a fusion product is preferably obtained by fusing the coding gene sequences for the antigen recognition regions VL and VH of the antibody to the gene sequences for a carrier molecule such as a surface protein of a phage or a capsid protein of a virus; this hybrid gene is then expressed in vitro. The method for isolating and cloning the gene sequences coding for the V regions of the antibody and their insertion into the DNA of other genes, e.g. in the gene sequence for the g3p phage protein and methods for expressing such a fusion protein are known to the expert (Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989): 3833-3837; Chiang et al., BioTechniques 7 (1989): 360-366; Winter, G., and Milstein, C., Nature 349 (1991): 293-299). The fusion products (on the surface of phages or viruses or in isolated form) can then be used like an antibody in the detection of the complexes.
Finally, the complexes in the cells to be examined can also be detected by hybridization of a DNA fraction of these cells with a DNA in accordance with the invention. The advantage of this method is that it is possible to distinguish with these DNA probes between cells of different differentiation degrees (fibroma and/or ascites tumor) and species (mouse or human).
Another subject matter of the invention is, hence, a method for the detection of human or animal cells having an unlimited proliferation or tumor-formation potential by hybridization of a DNA fraction of these cells with a DNA of the invention.
A cytoplasmic cell fraction which contains the complexes of the invention can be used as DNA fraction. It is, however, also possible to use nuclear, chromosomal DNA of the cells to be examined. Experience has shown that, as compared to cells with the capability of unlimited proliferation, nuclear chromosomal DNA of normal transient proliferating cells has, after restriction digestion and analysis of the restriction fragments in a southern blot, another pattern of hybridizing restriction fragments when hybridizing a DNA sequence in accordance with the invention. Usually, additional restriction fragments which hybridize with the probes indicate the presence of cells having an unlimited proliferation and tumor-formation potential.
The DNA sequences in accordance with the invention exhibit such a high sensitivity and specificity that these samples can also be used for hybridization with cells or tissue in situ. Only those cells are labeled in the cytoplasm that exhibit unlimited proliferation or tumor-formation potential. For this in situ hybridization it is possible to use both cells that can be cultured in vitro and cells obtained in biopsies or tissue sections of biopsies without requiring a cultivation step in order to replicate the cellular material.
Another preferred subject matter of the invention is, hence, a variant of the method of the invention where the hybridization is carried out as an in situ hybridization.
The sensitivity of the method is further increased by replicating the DNA of the complexes from the cells to be examined in the detection reaction enzymatically, especially with the aid of polymerase chain reaction according to methods that are known to the expert (Mullis and Fallona, Meth. Enzymol. 155 (1987): 335-350; Saiki et al., Science 239 (1988), 487-491).
A preferred subject matter of the invention is, hence, a special embodiment of the method of the invention where a nucleic acid is enzymatically replicated from a lysate of the cells to be examined or a DNA or RNA fraction of these cells with the aid of oligonucleotides which hybridize with a DNA of the invention.
Such an amplification is possible although the DNA protein binding is stable with respect to high temperatures (1 hour at 90xc2x0 C.), high salt concentrations (e.g. 6.9 mol/l cesium chloride) and with respect to proteinase K digestion (100 xcexcg/ml for 1 hour at 60xc2x0 C.).
The described method for detecting cells having an unlimited proliferation or tumor-formation potential is used for the detection of malignantant tumors where the cells of a biopsy, a tissue section, or cells or excreted complexes from body fluids of humans and animals are used as sample materials. The described method is, hence, suitable for detecting these cells. Moreover, when correspondingly modified, the described methods can also be used to isolate these cells. In this procedure, the described method is used particularly for identifying the desired cells in a certain fraction during the isolation procedure. It is possible to use antibodies in accordance with the invention and subsequently isolate the cells by means of fluorescence activated sorting (FACS).
The following examples explain the invention in greater detail in combination with the sequence protocols which show particularly preferred DNA sequences.